Henri Braat

Infliximab but not etanercept induces apoptosis in lamina propria T-lymphocytes from patients with Crohn’s disease

BACKGROUND & AIMS Steroid-refractory Crohns disease responds to therapy with the chimeric anti-tumor necrosis factor (TNF)-alpha antibody infliximab. Etanercept, a recombinant TNF receptor/immunoglobulin G fusion protein, is highly effective in rheumatoid arthritis but not in Crohns disease. Because both infliximab and etanercept are TNF-alpha-neutralizing drugs, we investigated the differences in TNF-alpha-neutralizing capacity and human lymphocyte binding and apoptosis-inducing capacity of both molecules. METHODS We used a nuclear factor kappaB reporter assay and a cytotoxicity bioassay to study TNF-alpha neutralization by infliximab and etanercept. Lymphocyte binding and apoptosis-inducing capacity was investigated using fluorescence-activated cell sorter analysis, annexin V staining, and cleaved caspase-3 immunoblotting using mixed lymphocyte reaction-stimulated peripheral blood lymphocytes (PBL) from healthy volunteers and lamina propria T cells from patients with Crohns disease. RESULTS Both infliximab and etanercept neutralized TNF-alpha effectively. Infliximab bound to activated PBL and lamina propria T cells, whereas binding of etanercept was equal to a nonspecific control antibody. Infliximab but not etanercept induced peripheral and lamina propria lymphocyte apoptosis when compared with a control antibody. Infliximab activated caspase 3 in a time-dependent manner, whereas etanercept did not. CONCLUSIONS Although both infliximab and etanercept showed powerful TNF-alpha neutralization, only infliximab was able to bind to PBL and lamina propria T cells and subsequently to induce apoptosis of activated lymphocytes. These data may provide a biological basis for the difference in efficacy of the 2 TNF-alpha-neutralizing drugs.

Increased expression of DC-SIGN+IL-12+IL-18+ and CD83+IL-12-IL-18- dendritic cell populations in the colonic mucosa of patients with Crohn's disease.

Dentritic cells (DC) as antigen‐presenting cells are most likely responsible for regulation of abnormal T cell activation in Crohns disease (CD), a chronic inflammatory bowel disease. Wehave analyzed the expression of activation and maturation markers on DC in the colon mucosa from patients with CD compared with normal colon, using immunohistochemical techniques. We found two distinct populations of DC present in CD patients: a DC‐specific ICAM‐3 grabbing non‐integrin (DC‐SIGN)+ population that was present scattered throughout the mucosa, and a CD83+ population that was present in aggregated lymphoid nodules and as single cells in the lamina propria. In normal colon the number of DC‐SIGN+ DC was lower and CD83+ DC were detected only in very few solitary lymphoid nodules. Co‐expression of activation markers and cytokine synthesis was analyzed with three‐color confocal laser scanning microscopy analysis. CD80 expression was enhanced on the majority of DC‐SIGN+ DC in CD patients, whereas only a proportion of the CD83+ DC co‐expressed CD80 in CD as well as in normal tissue. Surprisingly, IL‐12 and IL‐18were only detected in DC‐SIGN+ DC and not in CD83+ DC. A similar pattern of cytokine production was observed in normal colon albeit to a much lesser extent. The characteristics ofthese in‐situ‐differentiated DC markedly differ from the in‐vitro‐generated DC that simultaneously express DC‐SIGN, CD83 and cytokines.

Antibiotics with a selective aerobic or anaerobic spectrum have different therapeutic activities in various regions of the colon in interleukin 10 gene deficient mice

Background and aims: Multiple rodent models implicate resident intestinal bacteria in the pathogenesis of chronic immune mediated intestinal inflammation. Specific pathogen free (SPF) interleukin 10 gene deficient (IL-10−/−) mice develop colitis, which does not occur in the germ free (GF) state. We investigated whether broad or narrow spectrum antibiotics affect onset and progression of disease in various regions of IL-10−/− mice. Methods: Metronidazole, ciprofloxacin, vancomycin-imipenem (50 mg/kg/day), or water (control) was administered orally before (prevention) or two weeks after (treatment) colonisation of GF IL-10−/− mice with SPF bacteria. After four weeks, colonic histology scores and cytokine production by colonic explants were determined. Caecal and colonic contents were collected for quantitative bacterial analysis. Results: In the prevention study, all antibiotics decreased inflammation in the caecum and colon. However, in the treatment study, ciprofloxacin and vancomycin-imipenem decreased caecal inflammation, and reduced Escherichia coli and Enterococcus faecalis concentrations, whereas only vancomycin-imipenem lowered direct microscopic bacterial counts. In contrast, metronidazole and vancomycin-imipenem reduced colonic injury and eliminated anaerobic bacteria, including Bacteroides spp. Conclusions: Both narrow and broad spectrum antibiotics can prevent disease but treatment of established colitis is more selective. Ciprofloxacin is most effective in the treatment of caecal inflammation, metronidazole preferentially treats the colon, whereas vancomycin-imipenem definitively treats both regions. These results suggest that subsets of aerobic or anaerobic bacteria show regional differences in their capacity to mediate experimental colitis in IL-10−/− mice.

The prevalence of fat-soluble vitamin deficiencies and a decreased bone mass in patients with chronic pancreatitis

BACKGROUND/OBJECTIVES In chronic pancreatitis, malabsorption of fat is common due to loss of exocrine function. Consequently, these patients are at risk to acquire deficiencies of the fat-soluble vitamins, which may result in a decreased bone mineral density (BMD) and the development of osteopenia and osteoporosis. METHODS We prospectively enrolled all patients diagnosed with chronic pancreatitis, who visited our outpatient clinic between March and November 2011. Data were collected regarding demographic characteristics, symptoms, and pancreatic function. Serum concentrations of vitamins A, E, K, and D were determined, and BMD was assessed by means of bone densitometry. Results were analyzed according to pancreatic function status and enzyme use, and compared to reference data, when available. RESULTS Forty patients were included (43% female; mean age of 52). Alcohol abuse was the major cause of pancreatitis (50%). Twenty-eight patients were exocrine insufficient (70%), of whom 19 used pancreatic enzymes. Vitamin A, D, E, and K deficiencies were present in 3, 53, 10, and 63% of patients, respectively. Osteopenia and osteoporosis were observed in 45% and 10% of patients. A decreased BMD was more frequently observed than expected, based on reference data, even in exocrine sufficient patients. CONCLUSIONS Deficiencies of fat-soluble vitamins and a decreased BMD are frequently present in chronic pancreatitis, even in exocrine sufficient patients. Consequently, all patients with chronic pancreatitis should be routinely screened for fat-soluble vitamin deficiencies and a decreased BMD.

Interleukin-10-based therapy for inflammatory bowel disease

In recent years it has become clear that chronic inflammatory bowel disease (IBD), especially Crohn’s disease (CD), is caused by a loss of tolerance against the autologous bacterial flora of the intestine. Tolerance against the indigenous flora requires optimal recognition of antigens by pattern recognition receptors and the presence of important regulatory cells and cytokines. Interleukin-10 (IL-10) has a major role in the regulatory network of cytokines controlling mucosal tolerance, and it is, therefore, not surprising that this cytokine is proposed as a potent anti-inflammatory biological therapy in chronic IBD. This review will discuss the characteristics of IL-10, its immunoregulatory properties in mice and humans, and the use of IL-10 as a treatment for CD. The review will summarise the clinical studies that have taken place and discuss the lessons learned from these trials. Finally, the advantages and disadvantages of promising new strategies of IL-10 treatment, including gene therapy and the use of genetically modified bacteria, will be discussed. Both novel therapies have been shown to be successful in animal models of disease, and clinical testing is currently underway. The future goal of IL-10 treatment should be focused on mucosal delivery and remission maintenance instead of remission induction. In conclusion, it can be said that despite the disappointing results of IL-10 therapy so far, there is still enough rationale for the use of IL-10 as an anti-inflammatory biological treatment in chronic IBD.

Immunology of Crohn's disease

Abstract:  The immense microbiological load of the gastrointestinal tract poses a daunting challenge for the mucosal immune system: whereas it should tolerate the vast number of commensal bacteria, it should adequately attack pathogenic organisms. Millions of years of co‐evolution have produced an intricate system in which interactions between the endogenous flora and mucosal immune system manage to perform this difficult balancing act. When components of this interaction are defective, for instance by mutation, inflammatory bowel disease may result. In the present review, we comprehensively discuss the mucosal immune system in the context of Crohns disease (CD) and its genetic risk factors, describe the clinical management of the disease, and discuss how knowledge of the mucosal immune system may yield novel therapeutical avenues for dealing with this debilitating disease.

Prevention of experimental colitis by parenteral administration of a pathogen-derived immunomodulatory molecule

Background: Filamentous haemagglutinin (FHA) of Bordetella pertussis subverts host immune responses by inhibiting interleukin (IL)12 and enhancing IL10 production by macrophages and dendritic cells, and promoting the induction of regulatory T cells. Hypothesis: Injection of FHA would ameliorate disease in a T cell-dependent model of colitis via the induction of anti-inflammatory cytokines and regulatory T cells. Methods: Colitis was induced by injection of CD4CD45RBhigh naive T cells into severe combined immunodeficient (SCID) mice. Mice were treated with four subcutaneous injections of FHA or buffer alone. Results: Parenteral injection of FHA stimulated IL10 and/or transforming growth factor β production in local and mesenteric lymph nodes and Peyer’s patches of mice 2–6 h after administration. Compared with phosphate-buffered saline-treated mice, FHA-treated SCID mice had significantly (p<0.01) less weight loss, lower colon weight, less colon shrinkage and reduced inflammatory lesions. The therapeutic effect of FHA was associated with enhanced IL10 and reduced type 1 and type 2 T helper cytokine production by spleen cells. Finally, FHA also attenuated the symptoms of colitis in SCID mice transferred with CD4CD45RBhigh T cells from IL10-deficient mice. Conclusions: Our finding shows that FHA suppresses type 1 T helper and pro-inflammatory cytokines, and ameliorates disease activity in a chronic T cell-dependent model of colitis, an effect that was not dependent on IL10 production by T cells, but was associated with induction of anti-inflammatory cytokines in vivo. Having already been used as a pertussis vaccine component in children, FHA is a promising candidate for clinical testing in patients with Crohn’s disease.

Genetically Modified Lactococcus lactis for Delivery of Human Interleukin-10 to Dendritic Cells

Interleukin-10 (IL-10) plays an indispensable role in mucosal tolerance by programming dendritic cells (DCs) to induce suppressor Th-cells. We have tested the modulating effect of L. lactis secreting human IL-10 (L.  lactis IL-10) on DC function in vitro. Monocyte-derived DC incubated with L.  lactis IL-10 induced effector Th-cells that markedly suppressed the proliferation of allogenic Th-cells as compared to L. lactis. This suppressive effect was only seen when DC showed increased CD83 and CD86 expression. Furthermore, enhanced production of IL-10 was measured in both L.  lactis IL-10-derived DC and Th-cells compared to L. lactis-derived DC and Th-cells. Neutralizing IL-10 during DC-Th-cell interaction and coculturing L.  lactis IL-10-derived suppressor Th-cells with allogenic Th-cells in a transwell system prevented the induction of suppressor Th-cells. Only 130 pg/mL of bacterial-derived IL-10 and 40 times more exogenously added recombinant human IL-10 were needed during DC priming for the generation of suppressor Th-cells. The spatially restricted delivery of IL-10 by food-grade bacteria is a promising strategy to induce suppressor Th-cells in vivo and to treat inflammatory diseases.

Pancreatic cancer: Promise for personalised medicine?

Pancreatic cancer has an infaust prognosis and is the fourth commonest cause of cancer related death in men. Design of rational treatment has been hampered by lack of insight into the pathogenesis of the disease. Recently more insight has been gained into a number of crucial aspects of pancreatic carcinogenesis, in particular the cell types that can give rise to oncological transformation in the pancreas, different modes of interaction between transformed pancreatic cells and the stroma that fosters further disease progression, the need of the pancreatic tumour cells to overcome the pressure of immune surveillance and the various changes in intercellular biochemistry that tumour cells employ to both sustain chemoresistance and metastasis. Although still largely incomplete, this new knowledge opens novel avenues on more successful treatment of the disease through personalised medicine.

mTOR is a promising therapeutical target in a subpopulation of pancreatic adenocarcinoma

Pancreatic ductal adenocarcinoma (PDAC) remains a highly lethal disease, unusually resistant against therapy. It is generally felt that stratification of patients for personalized medicine is the way forward. Here, we report that a subpopulation of PDACs shows strong activation of the mTOR signaling cassette. Moreover, we show that inhibition of mTOR in pancreatic cancer cell lines showing high levels of mTOR signaling is associated with cancer cell death. Finally, we show using fine needle biopsies the existence of a subpopulation of PDAC patients with high activation of the mTOR signaling cassette and provide evidence that inhibition of mTOR might be clinically useful for this group. Thus, our results define an unrecognized subpopulation of PDACs, characterized by high activation of mTOR and show that identification of this specific patient group in the early phase of diagnosis is feasible.